The invention relates to a method for quantitatively and/or qualitatively detecting a specific binding substance in blood or a derived fraction thereof using the binding affinity between the specific binding substance and its co-reactant. Moreover a reaction component with peroxidase activity has been used by incubating a reaction mixture containing the specific binding substance and its co-reactant and by which the peroxidase activity in said reaction mixture or a fraction derived therefrom has been determined. The peroxidase activity is a measure of the quantity and/or presence of the specific binding substance. Also a test kit for carrying out said method is part of the invention. Such a method to demonstrate a peroxidase activity has already been described in Dutch Patent 154,600.
As carrier material in said method has often been used the wall of a small well in a microtitration plate to which antibodies are bonded. The blood to be investigated or a fraction derived therefrom, such as serum, is introduced into the small wells of the microtitration plate. The bonded antibody is able to react with the antigen present in the blood or a derived fraction thereof, as a result of which said antigen is bonded. In order to be able to detect whether an immunochemical reaction has taken place, subsequently one can use marked antibodies which are directed against the antigen to be determined. Said marked antibodies will react with the antibody-antigen complex formed already on the solid phase. The marked antibodies consist of antibodies to which an enzyme is bonded, preferably a peroxidase-enzyme. In the next step, a colourless solution (substrate+chromogen) is added. The enzyme is able to convert this solution into a coloured compound. The intensity of the colour depends on the quantity of enzyme which is proportional to the quantity of bound antigen. Said colour can be altered by adding a stop reagent. The method thus described of determining an antigen in blood or a derived fraction thereof is a method of the sandwich type. Other immunochemical methods, such as an inhibition reaction and a competition reaction, can also be used. As suitable enzymes one can use, inter alia, alkaline phosphatase, urease and a previously mentioned peroxidase, preferably Horse Radish Peroxidase (HRP). The HRP catalyses the conversion of the substrate, a peroxide compound, in water. The electrons needed for this reaction may be supplied by chromogenic substances such as tetramethylbenzidine (TMB). The TMB is converted as a result into coloured complexes, by which the enzyme action is demonstrated. The colour formed can either be detected visually or the enzyme action can be determined in an analytically suitable manner.
In examining blood for the presence of antigens or antibodies, it is usual that the serum containing the antigens or antibodies to be determined has been separated from the blood cells prior to the determination.
In practice it emerges that an undesired, increased colour intensity is often produced, in particular in carrying out an enzyme immunoassay (EIA) when a peroxidase being used as enzyme. This phenomenon is also described as partial coloration.
It will be clear that this phenomenon of partial coloration will lead to undesired results and conclusions. In this connection one can mention so-called AIDS antibody tests. In these tests sera of blood donors are tested routinely in an enzyme immunoassay for the presence of antibodies directed against the AIDS virus.